Mechanisms of antibiotic resistance in Burkholderia pseudomallei: implications for treatment of melioidosis

Abstract

Burkholderia pseudomallei is the etiologic agent of melioidosis. This multifaceted disease is difficult to treat, resulting in high morbidity and mortality. Treatment of B. pseudomallei infections is lengthy and necessitates an intensive phase (parenteral ceftazidime, amoxicillin-clavulanic acid or meropenem) and an eradication phase (oral trimethoprim-sulfamethoxazole). The main resistance mechanisms affecting these antibiotics include enzymatic inactivation, target deletion and efflux from the cell, and are mediated by chromosomally encoded genes. Overproduction and mutations in the class A PenA β-lactamase cause ceftazidime and amoxicillin-clavulanic acid resistance. Deletion of the penicillin binding protein 3 results in ceftazidime resistance. BpeEF-OprC efflux pump expression causes trimethoprim and trimethoprim-sulfamethoxazole resistance. Although resistance is still relatively rare, therapeutic efficacies may be compromised by resistance emergence due to increased use of antibiotics in endemic regions. Novel agents and therapeutic strategies are being tested and, in some instances, show promise as anti-B. pseudomallei infectives.

Figure 1. Bacterial antibiotic resistance mechanisms

Bacterial antimicrobial resistance mechanisms are multifaceted, ranging from: exclusion of drug molecules (blue spheres) from the bacterial cell by physicochemical constraints (e.g., porins or lipopolysaccharide); efflux from the cell via active transport mechanisms; enzymatic inactivation, either in the form of substrate cleavage or chemical modification (M = acetylation, adenylation or phosphorylation); target site alteration or, rarely, target deletion; metabolic bypass by substitution of a susceptible enzyme or pathway with a resistant enzyme or pathway; target overproduction by either increased transcription or gene multiplication; and drug sequestration by specific binding proteins akin to immunity proteins. Details about individual resistance mechanisms and specific examples are provided in the text. Bacteria often employ different resistance mechanisms that act synergistically, for instance, efflux and exclusion, to achieve high-level resistance [26].

Figure 2. Burkholderia pseudomallei PenA and locations of mutations leading to clinically significant antibiotic resistance

Positions of conserved regions and mutations are numbered according to the Ambler scheme [69]. Susceptibilities to clinically significant β-lactam antibiotics range from 1.5 to 3 µg/ml (wild-type) [33,34,36,64], ≥256 µg/ml (C69Y) [33,34,64] and 24 to 64 µg/ml (P167S) [36,64] for ceftazidime, and 3 to 8 µg/ml (wild-type) and 16 to 32 µg/ml (P72F) for amoxicillin–clavulanic acid [64,67]. By comparison, the meropenem MICs in all isolates are not significantly affected and range from 0.75 to 1.5 µg/ml [36,64]. Current susceptibility breakpoints are ≤8 µg/ml for ceftazidime, ≤8 µg/ml for amoxicillin and ≤4 µg/ml for clavulanic acid.

Figure 3. Summary of Burkholderia pseudomallei resistance mechanisms compromising therapy with clinically significant antibiotics

Enzymatic inactivation and efflux from the cell are mechanisms that compromise the use of antibiotics employed in intensive and eradication phase therapy. The CEF and AMX targets are located in the periplasm. There is experimental evidence that CEF and other β-lactams permeate the outer membrane through porins. Periplasmic β-lactams, such as AMX, are (A) inactivated by the wild-type PenA class A β-lactamase, (B) unless its activity is inhibited by CLA. Although wild-type PenA hydrolyzes CEF to some extent, it does not confer clinically significant resistance to CEF. (C) Some mutant PenA (PenA^mt) derivatives catalyze CEF hydrolysis and others (not illustrated) are refractory to CLA inhibition. Antibiotics such as DOX, TMP and TMP–SMX have cytoplasmic targets and are extruded to the extracellular milieu by the multicomponent BpeEF–OprC resistance nodulation and cell division efflux pump. As resistance nodulation and cell division pumps extrude substrates acquired from the periplasmic space, the listed antibiotics are most likely extruded either during their transit into the cell or after extrusion to the periplasm from the cytoplasmic space via an unknown mechanism. AMX: Amoxicillin; CEF: Ceftazidime; CLA: Clavulanic acid; DOX: Doxycycline; SMX: Sulfamethoxazole; TMP: Trimethoprim.